The illnesses afflicting men and women who served in the Persian Gulf military conflict during 1990-1991 remain ill-defined. A constellation of symptoms including fatigue, rashes, headaches, arthralgias, myalgias, diarrhea, memory loss, autoimmune thyroid disease, increased allergies and sensitivities to environmental elements, and neurological abnormalities collectively referred to as Gulf War Syndrome (GWS) have been described (Grady, et al. Arch. Int. Med. 158: 367-371, 1998; Persian Gulf Veterans Coordinating Board. Arch. Int. Med. 155: 262-268, 1995; Haley, et al. J.A.M.A. 277: 231-237, 1997). While GWS patients do not in general suffer from classic rheumatic diseases, the signs and symptoms are reminiscent of atypical connective tissue diseases such as fibromyalgia, chronic fatigue syndrome, and the process associated with exposure to silicone breast implants (SBI). Serological abnormalities including hypergammaglobulinemia and abnormal serum proteins have been reported in 45% of GWS patients (Grady, et al. Arch. Int. Med. 158: 367-371, 1998).
Hundreds of explanations for GWS have been proposed. In 1994, the U.S. Secretary of Defense and the Secretary of Veterans Affairs asked the Center for Disease Control and Prevention to conduct an official scientific study exploring possible causes of GWS. The study was aimed at organizing reported symptoms into a defined case, characterizing clinical features, and evaluating risk factors. The results are described by Fukuda, et al (J.A.M.A. 280: 981-988, 1998). Fukuda et al, assessed a population of Gulf War Veterans with respect to many of the proposed explanations for GWS. The study included assessment of physical symptoms; blood, urine, and stool analysis; and
serological assays. Tests were conducted to detect the presence of various viruses, bacteria, mycoplasm, and parasites. Serum was tested for yellow fever, dengue, Sindbis, West Nole, and phlebotomus fever viruses (Naples and Sicilian); Toscana, Karimbad, and Isfahan viruses; Rickettsia typhi and Rickettsia rickettsii; Coxiellla burnetii; Ehrlichia chaffeensis; Leishmania tropica and Leishmania donovani; Toxoplasma gondii; Schistosoma mansoni and Schistosoma haematobium; Strongyloides stercoralis; Helibacter pylori; Clostridium botulinum; and Bacillus anthracis. Stool specimens were tested for red and white blood cells; ova and parasites of Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli, and microsporidia; enteroviruses; and bacteria strains of Salmonella, Shigella, Yersinia, Campylobacter, and Escherichia coli (0157:H7). While this study is considered an official and comprehensive report on GWS, no attempt was made to assess alternative explanations, such as adjuvant's disease.
Other theories, implicating exposure to chemical and biological agents as a cause of GWS, have been the subject of numerous studies. These studies, however, neither conclusively identify a causative agent for the disease nor properly explain the pathology observed in GWS cases.
The Persian Gulf Veterans Coordinating Board has addressed the possibility of exposures to chemical and biological agents. The Board, however, attempted to account for these illnesses without defining a molecular pathology (Persian Gulf Veterans Coordinating Board. Arch. Int. Med. 155: 262-268, 1995).
Haley grouped reported symptoms into seven different syndromes based upon possible exposure to various chemicals present at the time of the Persian Gulf Conflict (Haley, et al. J.A.M.A. 277: 231-237, 1997). While this study does attempt to categorize possible causes of the disease, it fails to explain the GWS pathology observed in the veterans. Furthermore, the study did not outline the pathology one would expect to observe in those exposed to the various chemical agents.
Abou-Donia examined acute toxicity of pyridostygmine bromide and organophosphates in chickens (Abou-Donia, et al. J. Tox. Environ. Health. 48: 35-56, 1996). The study compared chickens suffering from acute toxicity to the condition presented by GWS patients. However, since many Gulf War veterans did not develop GWS until months or years after the military conflict, an animal model which employs a rapid acute response to mimic the symptoms of GWS may not be appropriate. As a result, the study examining acute toxicity in chickens may not be relevant to a human disease characterized by a delayed onset. Furthermore, the study did not fully reflect the molecular pathology observed in actual GWS cases.
While chemical toxicity may mimic some of the GWS symptoms, it cannot account for all of them. For instance, exposure to organophosphates has never been associated with the development of immunological abnormalities (Vial, et al. J. Tox. Environ. Health. 48: 215-229, 1996). Chronic organophosphate pesticide intoxication in two mammalian species neither interfered with cell-mediated immunity, nor induced autoimmunity (Jha, et al. Acta. Vet. Hung 38: 55-60, 1990). Clearly, this is distinct from GWS, which displays a multitude of symptoms linked to immune dysfunction.
An additional example involving chemical agents is provided by the large numbers of individuals exposed to sarin in the Japanese subway Mar. 20, 1995. Acetylcholinesterase levels were monitored and observed to return to normal levels in all patients by three months following exposure. Some subclinical miosis and neuropathy were present 30 days after exposure, but these disappeared after a year (Morita, et al. Lancet. 346: 290-3, 1995). Tissue distribution of sarin and its metabolites, as well as the time course of detoxification following exposure is known (Little, et al. Toxicol. Appl. Pharmacol. 83: 412-419, 1986). The physiological effects are immediate and the pathology is well documented. In no case has there been reports of any delayed onset of autoimmune disorders.
No exposures to chemical agents which required medical treatment were documented by U. S. military personnel. Such exposures typically require the use of ventilatory or circulatory supports, or atropine and anticonvulsants to combat the onset of acute symptoms. Low dose effects were also not observed (Dr. Bernard Rostker, Pentagon, personal communication; Col. Edward Koenigsburg, USAF, Persian Gulf Investigation, Falls Church, Va., personal communication). Such effects have been studied in primates and generally abate within 24 hours of the exposure (Wolthuis, et al. Pharmacol. Biochem. Behav. 51: 443-456,1995).
Others have proposed that GWS is a type of post-traumatic stress syndrome (Hyams, et al. Ann. Int. Med. 125: 398-405, 1996). It is difficult, however, to reconcile this hypothesis with the symptoms manifest in afflicted individuals.
An additional explanation has been outlined in which GWS results from a dysregulation of the immune system. (Hyams, et al. Ann. Int. Med. 125: 398-405, 1996). The GWS patients suffer from various symptoms similar to those having autoimmune diseases, but cannot be diagnosed with a "classic" rheumatic disease. Gulf War veterans and attendant civilian personnel received a variety of immunizations in preparation for possible deployment to the Persian Gulf theater (David, et al. Br. Med. J 314: 239-240, 1997). It has been suggested that GWS may result from an imbalance in the immune system. It was hypothesized that the imbalance may be due to an adverse reaction to a vaccination.
It was noted in some patients that the onset of illness occurred within weeks of receiving a full complement of immunizations. These individuals displayed symptoms of GWS soon after vaccination and were not deployed. Other individuals were deployed, but returned home before the start of the war because of severe joint and muscle pain, as well as neurological problems. Additional personnel from the Gulf War became ill years later. These individuals, however, report the same symptoms as those who became ill only weeks after their vaccinations. The variability in the onset of disease symptoms, as well as differences in their severity, may be due to individual immune responses. Such variability is reportedly regulated at a genetic level involving the histocompatibility complex (Madzhidov, et al. Biull. Eksp. Biol. Med. 102: 74-76, 1986; Lorentzen, et al. Transplant. Proc. 27:1532-1534, 1995).
A possibility exists that the immunizations administered to all personnel involved in the Gulf War may be linked to the etiology of GWS. The immunizations administered typically comprised an antigen and an immunological adjuvant. The adjuvants function to boost the protective effect of the immunization by eliciting a stronger immune response against the antigen. The adjuvants are capable of stimulating the immune system's cell-mediated and humoral responses against the antigen being administered. Cases have been reported, however, where the adjuvants cause a more generalized and indiscriminate stimulation of the immune system. This can disrupt the balance of self-regulatory mechanisms within the immune system and lead to autoimmune disease (Kleinau, et al. Scand. J. Rheumatol. 101: 179-181, 1995; Madzhidov, et al. Biull. Eksp.
Biol. Med. 102: 74-76, 1986; Lorentzen, et al. Transplant. Proc. 27: 1532-1534, 1995).
The adjuvant most commonly used is alum, which is most often provided as aluminum hydroxide, Al(OH).sub.3. This adjuvant appears to induce its effect by slowing the release of antigen from an antigen/adjuvant complex, as well as by other immunostimulatory properties such as attraction of immunocompetent cells to the injection site. In preparing vaccines, antigens are physically precipitated with hydrated insoluble salts of aluminum hydroxide or aluminum phosphate. The resulting composition is further processed and provided in a convenient form for administration. Alum is the only adjuvant approved by the Food and Drug Administration (FDA) for human use. It has been given to millions of people world-wide in various vaccines with no reports of significant problems (Warren, H. S., et al. Ann. Rev. Immunol. 4:369-388, 1986).
Although not approved by the FDA for use in humans, squalene has been used as an adjuvant in experimental vaccines against a variety of pathogens including Bacillus anthracis (Ivans, et al. Vaccine. 13: 1779-1783, 1995), Plasmodium falciparum, (Hoffman, et al. Am. J Trop. Med. Hyg. 51: 603-612, 1994), and herpes simplex virus (Burke, et al. J. Inf. Dis. 170: 1110-1119, 1994). The effectiveness of adjuvants has been shown to correlate with toxicity. This toxicity typically manifests with the onset of autoimmune disease symptoms (Koga, et al. Microbiol. Immunol. 30:717-723, 1986; Zamma, et al. Infect. Immun. 39: 1291-1299, 1983).
A study using squalene as an adjuvant in an influenza vaccine reported moderate to severe local and systemic immune reactions in humans (Keutek, et al. Vaccine. 11: 909-913, 1993). The participants reported induration, erythema, lymphadenopathy, fever, chills, nausea, and dizziness lasting for several days.
Another squalene-containing vaccine was used with gp120 in a candidate human immunodeficiency virus (HIV) vaccine. In this case, induction of severe systemic and local reactions was reported in 15 of the 30 participants (Keefer, et al. AIDS Res. Hum. Retro. 12: 683-693, 1996). Similarly, in a study of a simian immunodeficiency (SIV) vaccine in macaques, squalene was used as an adjuvant. The animals in this study developed anti-human-cell antibodies and various other autoimmune-like symptoms (Vaslin, et al. Int. Conf. AIDS 8(2):A42, 1992).
Several adjuvants have reportedly produced autoimmune diseases in experimental models. Adjuvant-induced arthritis is a well-characterized autoimmune disease which can be induced in rats and other species (Kleinau, et al. Scand. J. Rheumatol. 101: 179-181, 1995; Madzhidov, et al. Biull. Eksp. Biol. Med. 102: 74-76, 1986; Lorentzen, et al. Transplant. Proc. 27: 1532-1534, 1995). The disease process in these cases is complex, often affecting multiple organ systems. For example, cachectic syndrome (Rofe, et al. Agents Actions. 42: 60-62, 1994) and testicular dysfunction (Clemons, et al. J. Androl. 10: 419-424, 1989) have been associated with adjuvant-induced arthritis. Uveitis, a T-cell mediated intraocular inflammatory disease, can also be induced by adjuvants (Petty, et al. J. Rheumatol. 16: 400-405, 1989). Additional reports have shown that various neurological disorders, commonly manifest in patients with autoimmune diseases, may be the result of immunological dysfunction (Rogers, et al. Molecular Med. Today. 2: 76-81, 1996; Tekin, et al. Dementia 7: 91-94, 1996; Honnorat, et al. Arch. Neurol. 52: 462-468, 1995; Wucherpfennig, et al. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 68: 105-116, 1990; Cross, et al. Am. J. Pathol. 139: 1401-1409, 1991; Bansal, et al. J. Clin. Pathol. 47: 300-302, 1994; McNicholl, et al. J. Rheumatol. 21: 1061-1066, 1994; Zanone, et al Diabetologia. 36: 564-569, 1993; Moll, et al. Neurology. 43: 2574-2581, 1993).
GWS may also be a form of "adjuvants disease." It has been hypothesized that GWS may be caused by squalene administered in a vaccine to many Gulf War personnel. This theory, however, has been discounted by government and military officials. A report, prepared by the U.S. Army Medical Research and Material Command in 1996, discusses the squalene theory. "The basic hypothesis and supporting evidence presented are flawed or inaccurate. . . . Available information strongly argues against [this] hypothesis." The report goes on to outline seven reasons why the squalene theory is implausible or wrong (Rodriquez, "Sickness and Secrecy," Insight on the News. Aug. 25, 1997, pg. 9-10).
In addition to this rejection by the military and government, the squalene theory has been largely ignored by the scientific community. As discussed above, nearly all of the scientific studies investigating the causes of GWS have focused on chemical and biological agents (e.g. Fukuda, et al. J.A.M.A. 280: 981-988, 1998; Haley, et al. J.A.M.A. 277: 231-237, 1997; Persian Gulf Veterans Coordinating Board. Arch. Int. Med. 155: 262-268, 1995; Abou-Donia, et al. J. Tox. Environ. Health. 48: 35-56, 1996; Hyams, et al. Ann. Int. Med. 125: 398-405, 1996). These scientific studies have not adequately addressed the role of squalene in the etiology of GWS. Yet many close parallels exist between the symptoms of GWS and those observed in "adjuvants disease." Clearly, rejection of the squalene theory is premature. Thus, despite its dismissal by many scientific, governmental, and military organizations, the squalene theory is not adequately understood and warrants further study. More information is needed to discern the role that this adjuvant may play in the onset of GWS symptoms.
Understanding GWS at a molecular level is a first step towards identifying its cause and providing a successful treatment. To make this possible, diagnosis of patients with GWS needs to be more reliable. Reliable diagnosis is currently difficult given the wide array of symptoms associated with the disease. Thus, there exists a need in the art for new methods to aid in the diagnosis of GWS.